U.S. Pat. Nos. 6,232,523; 6,235,967; 6,235,968; and 6,251,384, as well as PCT applications PCT US 98/08457 and PCT US 00/00243, all of which are incorporated herein by reference, describe techniques for labeling tumors and their metastases using fluorescent markers, especially green fluorescent protein (in various colors) as well as detecting angiogenesis using this type of labeling, optionally along with a contrast dye. According to the descriptions of these documents, it is possible to detect tumor progression, angiogenesis, and metastasis by excising the tumor tissue, or, perhaps more benevolently, in real time by following the course of the condition through whole body imaging. The imaging of this condition through the skin has been successful, but, not surprisingly, is somewhat limited in sensitivity due to scattering through the skin. Others have attempted to overcome the lack of sensitivity by invasive techniques which are of only modest success. For example, Brown, E B, et al., Nat Med, (2001) 7:864-868 used a dorsal skin chamber in mice with two photon confocal microscopy. Such an approach is limited to ectopic primary tumors. Naumov, G N, et al., J Cell Sci (1999) 112:1835-1842 described intravital microscopy of GFP-expressing tumors on exteriorized organs. This had the effect of serious morbidity. Subcutaneous windows of semitransparent materials are described by Siancio, S J, et al., J Surg Res (2000) 92:228-232. All of these methods suffer from serious disadvantages, not the least of which is that they are not suitable for repetitive measurement and cause serious harm to the model, thus distorting the results, as well as being capable only of limited time duration.
It is has now been found that by providing a simple skin flap, which can be opened and then resutured, an enhanced sensitivity, permitting observation of as little as a single cell, can be obtained while maintaining the subject in observable condition. The technique is minimally invasive and thus permits observation of tumor progression, angiogenesis, and metastasis with great precision often to a single cell level. Since the skin flap technique is relatively harmless, observation at later stages in the development of the tumor and its metastasis is possible. The intact animal is maintained. The ability to observe over long time periods also permits detection of dormant walls.
A report of this work was published by the present applicants in March of 2002: Yang, M., et al., Proc Natl Acad Sci (2002) 99:3824-3829. The contents of this publication are incorporated herein by reference.